The invention relates to cell culturing methods and devices, and their use in organ, e.g., liver, assist systems, and methods of making and using such devices.
Approximately 30,000 Americans die each year from liver diseases. Although liver transplantation has a survival rate in excess of 65%, many prospective recipients die while awaiting a donor. Attempts to develop extracorporeal devices and systems for liver replacement, such as microcarriers and hollow-fiber bioreactors, have shown only limited success. Some of these liver assist devices (LAD) have also been referred to as extracorporeal bioartificial livers (BAL). See, e.g., Uchino et al., ASAIO Transactions, 34(4):972-977 (1988); Taguchi et al., Artificial Organs, 20(2):178-185 (1996); Hu et al., U.S. Pat. No. 5,605,835; and Kelly, U.S. Pat. No. 5,290,684.
A BAL is an extracorporeal device used to replace liver function on a temporary basis. One embodiment of a BAL utilizes planar-configured cultures of one or more cell types. A BAL can support patients awaiting transplantation and can be used to stabilize patients during periods of recovery from fulminant hepatic failure.
The invention is based on the discovery that if the flow patterns and velocity of the liquid medium flowing between the plates in a flow-through cell culturing device are carefully controlled, the device can be used to culture cells at high levels of mass transport of nutrients, oxygen, and waste products, yet low levels of shear stress, which can limit cell survival and function. The invention is further based on the discovery that oxygenation of the cells can be further enhanced by separating the flow of culture medium and gas by a gas-permeable, liquid-impermeable membrane, thereby enabling low media flow rates to provide low shear stress, and high gas flow rates to provide a high level of oxygen to the cells.
Oxygen is delivered to cells by an oxygenated fluid. An oxygenated fluid can be gases or liquids and can include pure oxygen gas, air, oxygen-enriched air, and liquids that can be highly oxygenated.
The new flow-through cell culturing devices can thus be used to culture hepatocytes and fibroblasts for extended periods of time and with high levels of cell function in organ assist systems, such as hepatic function in liver assist systems.
The invention also includes new culturing plates for use in the flow-through culturing devices, and methods of manufacturing these plates.
In general, the invention features a flow-through cell culturing device including a housing with an inlet and an outlet; a first plate arranged within the housing; and a second plate arranged within the housing substantially in parallel with the first plate to create a chamber therebetween having a height of between about 25 and 500 microns, e.g., 50 to 100, 200, or 400 microns, wherein the chamber has a fluid entry and a fluid exit positioned such that fluid entering the housing through the inlet flows through the fluid entry of the chamber, flows through the chamber, exits the chamber through the fluid exit, and flows out of the housing through the outlet. The device can also include one or more cells, e.g., cultured, preserved (e.g., cryopreserved or dried), or freshly isolated hepatocytes or other cells, seeded onto a plate.
The device can further include a first manifold controlling the flow of liquid from the housing inlet directly to the fluid entry of the chamber; and a second manifold controlling the flow of liquid from the fluid exit of the chamber to the housing outlet. In this device, each plate can include a hole which serves as a fluid entry for the chamber below the plate, and as a second fluid exit from the chamber above the plate. The device can include any number of plates, all arranged within the housing in parallel, stacked one on top of the other.
Each plate in the device can be associated with at least three spacer elements located at three points in a plane on the plate, wherein all spacer elements have the same height. The plates can be made of, e.g., glass, polymethylmethacrylate, or polycarbonate. The spacer elements can be formed from the same or different material as the plates. For example, the spacer elements can be polymethylmethacrylate (PMMA), UV-cured acrylate adhesives, visible light curable adhesives, or polyurethane.
In another aspect, the invention features a flow-through cell culturing device including a housing with an inlet and an outlet; a first plate arranged within the housing; a second plate arranged within the housing substantially in parallel with the first plate to create a chamber therebetween; and a gas-permeable, liquid-impermeable membrane arranged between the first and second plates to create first and second compartments within the chamber, the first compartment having a height of about 5 microns to 5.0 millimeters, and the second compartment having a height of between about 25 and 500 microns, e.g., 50 to 100 or 200 microns, wherein the first compartment has a gas entry and a gas exit, and the second compartment has a fluid entry and a fluid exit positioned such that fluid entering the housing through the inlet flows through the fluid entry of the second compartment, flows through the second compartment, exits the second compartment through the fluid exit, and flows out of the housing through the outlet, and wherein the first compartment and the second compartment are not in fluid communication. The device can include one or more cells seeded onto the plate.
The gas-permeable, liquid-impermeable membrane can comprise polyurethane, polyolefin, polyethylene, polypropylene, polyvinylidene fluoride, polystyrene, nylon, silicone rubber, or mixtures or copolymers thereof. The device can include a substrate to support the membrane. The device can include additional plates and membranes arranged in a stack of alternating plates and membranes.
In another aspect, the invention features a method of microfabricating one or more uniform spacer elements on a plate, by depositing one or more separate volumes of a liquid polymer on a surface of the plate; curing the polymer to form one or more solid spacer elements; and machining the solid spacer elements to obtain a uniform height for each spacer element relative to the plate surface. The liquid polymer can be cured by exposure to UV light, air, or a catalyst. The spacer elements can be machined to a uniform height of between 10 and 200 microns, e.g, 50 microns. The liquid polymer can be, e.g., acrylate, siloxane, polyurethane, or epoxy resin.
The invention also features a method of microfabricating one or more uniform spacer elements on a plate by impressing a probe, e.g., a heated probe, into the surface of the plate with a sufficient force to form one or more microindentations with concomitant one or more micro-elevations; and machining the micro-elevations to obtain a uniform height for each micro-elevation relative to the plate surface, thereby forming the uniform spacer elements. The machining can include compressing top edges of the micro-elevations to obtain a uniform height for each micro-elevation relative to the plate surface, or removing top edges of the micro-elevations to obtain a uniform height for each micro-elevation relative to the plate surface.
In another aspect, the invention features an organ, e.g., liver, assist system including one or more of the new flow-through cell culturing devices described herein, a first conduit for conducting plasma from a patient to the housing inlet; a second conduit for conducting plasma from the cell culturing device to the patient; and a pump for moving plasma through the conduits and cell culturing device. The system can further include a plasma separator to remove blood cells from whole blood to provide plasma that is passed through the cell culturing device, and/or a bubble trap, to remove bubbles from the plasma in the first conduit prior to entering the cell culturing device.
The invention also includes a method of filtering a bodily fluid, e.g., blood or blood plasma, by seeding a cell culturing device as described herein with hepatocytes and/or fibroblasts or other cells; introducing the bodily fluid into the inlet of the device; and allowing the bodily fluid to flow through the device and exit through the outlet, thereby filtering the bodily fluid. The device can be seeded with 2 to 25 billion hepatocytes, e.g., porcine, human, bovine, ovine, equine, or murine hepatocytes. The bodily fluid can be introduced into the inlet at a flow rate of 0.05 to 400 ml/minute. The bodily fluid can be passed through a filter prior to introducing the bodily fluid into the inlet of the device.
In another embodiment, the invention features a modular device including a plurality of units, a fluid inlet conduit, and a fluid outlet conduit. Each unit includes a plate and a gas-permeable, liquid-impermeable membrane, e.g., with a substrate, arranged substantially in parallel with the plate and connected to the plate by an exterior wall to create a chamber therebetween having a height of between about 25 and 500 microns, wherein the chamber has a fluid entry connected to the fluid inlet conduit (e.g., via a valve) and a fluid exit connected to the fluid outlet conduit and positioned such that fluid entering the chamber flows through the chamber, exits the chamber through the fluid exit, and flows out of the modular device through the outlet. This modular device can be enclosed in a sealed container having a gas entry and gas exit.
The xe2x80x9cdead volumexe2x80x9d of a given device or system is the total liquid volume of the device including all fluid conduits and pumps that are part of the extracorporeal system. There is a limitation in this dead volume in a liver assist system. A large dead volume would cause dilution of the desired compounds produced by the hepatocytes within the liver assist system. The bioactivity of these compounds relies on a minimal critical concentration; therefore, dilution would compromise device performance.
When two plates are substantially in parallel, the plates have a distance between them that varies less than 20 percent from one end of the plates to the other. When two plates have a uniform distance between them, the distance does not vary by more than 10 percent from one end of the plates to the other.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The new flow-through cell culturing devices and liver assist systems provide numerous advantages. The new devices allow various cells to be cultured with high levels of mass transport and oxygenation while avoiding detrimental shear stress normally associated with high levels of mass transport. As a result, even shear-sensitive cells such as hepatocytes can be cultured for extended periods of time at high levels of function. This allows the new flow-through cell culturing devices to be used in organ, e.g., liver, assist systems.
In addition, the new cell culturing devices have an extremely small dead volume, which is important in organ assist systems, because it minimizes the amount of plasma or blood that is outside the body at any time, and also reduces dilutional effects.
The new methods of manufacturing the plates in the cell culturing devices are simple and economical, yet highly precise to provide the necessary control over flow patterns and dead volume in the new devices. Moreover, the new microindented spacers introduce no additional material to the biocompatible culture plates, and thus are inherently biocompatible.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.